What controls the length of noncoding DNA?

Several recent studies of genome evolution indicate that the rate of DNA loss exceeds that of DNA gain, leading to an underlying mutational pressure towards collapsing the length of noncoding DNA. That such a collapse is not observed suggests opposing mechanisms favoring longer noncoding regions. The presence of transposable elements alone also does not explain observed features of noncoding DNA. At present, a multidisciplinary approach--using population genetics techniques, large-scale genomic analyses, and in silico evolution--is beginning to provide new and valuable insights into the forces that shape the length of noncoding DNA and, ultimately, genome size. Recombination, in a broad sense, might be the missing key parameter for understanding the observed variation in length of noncoding DNA in eukaryotes.

Coding sequence evolution.

Dramatic progress has been made in the past ten years in the development of statistical and experimental techniques for investigating features of molecular evolution. Applied to coding regions, these techniques have produced remarkable advances in our understanding of selection for codon usage but, ironically, have had little impact on our understanding of protein evolution. That may be about to change.

The Hill-Robertson effect: evolutionary consequences of weak selection and linkage in finite populations.

The 'Hill-Robertson (HR) effect' describes that linkage between sites under selection will reduce the overall effectiveness of selection in finite populations. Here we discuss the major concepts associated with the HR effect and present results of computer simulations focusing on the linkage effects generated by multiple sites under weak selection. Most models of linkage and selection forecast differences in effectiveness of selection between chromosomes or chromosomal regions involving a number of genes. The abundance and physical clustering of weakly selected mutations across genomes, however, justify the investigation of HR effects at a very local level and we pay particular attention to linkage effects among selected sites of the same gene. Overall, HR effects caused by weakly selected mutations predict differences in effectiveness of selection between genes that differ in exon-intron structures and across genes. Under this scenario, introns might play an advantageous role reducing intragenic HR effects. Finally, we summarize observations that are consistent with local HR effects in Drosophila, discuss potential consequences on population genetic studies and suggest future lines of research.

Synonymous substitutions in the Xdh gene of Drosophila: heterogeneous distribution along the coding region.

The Xdh (rosy) region of Drosophila subobscura has been sequenced and compared to the homologous region of D. pseudoobscura and D. melanogaster. Estimates of the numbers of synonymous substitutions per site (Ks) confirm that Xdh has a high synonymous substitution rate. The distributions of both nonsynonymous and synonymous substitutions along the coding region were found to be heterogeneous. Also, no relationship has been detected between Ks estimates and codon usage bias along the gene, in contrast with the generally observed relationship among genes. This heterogeneous distribution of synonymous substitutions along the Xdh gene, which is expression-level independent, could be explained by a differential selection pressure on synonymous sites along the coding region acting on mRNA secondary structure. The synonymous rate in the Xdh coding region is lower in the D. subobscura than in the D. pseudoobscura lineage, whereas the reverse is true for the Adh gene.

The correlation between intron length and recombination in drosophila. Dynamic equilibrium between mutational and selective forces.

Intron length is negatively correlated with recombination in both Drosophila melanogaster and humans. This correlation is not likely to be the result of mutational processes alone: evolutionary analysis of intron length polymorphism in D. melanogaster reveals equivalent ratios of deletion to insertion in regions of high and low recombination. The polymorphism data do reveal, however, an excess of deletions relative to insertions (i.e., a deletion bias), with an overall deletion-to-insertion events ratio of 1.35. We propose two types of selection favoring longer intron lengths. First, the natural mutational bias toward deletion must be opposed by strong selection in very short introns to maintain the minimum intron length needed for the intron splicing reaction. Second, selection will favor insertions in introns that increase recombination between mutations under the influence of selection in adjacent exons. Mutations that increase recombination, even slightly, will be selectively favored because they reduce interference among selected mutations. Interference selection acting on intron length mutations must be very weak, as indicated by frequency spectrum analysis of Drosophila intron length polymorphism, making the equilibrium for intron length sensitive to changes in the recombinational environment and population size. One consequence of this sensitivity is that the advantage of longer introns is expected to decrease inversely with the rate of recombination, thus leading to a negative correlation between intron length and recombination rate. Also in accord with this model, intron length differs between closely related Drosophila species, with the longest variant present more often in D. melanogaster than in D. simulans. We suggest that the study of the proposed dynamic model, taking into account interference among selected sites, might shed light on many aspects of the comparative biology of genome sizes including the C value paradox.

The correlation between synonymous and nonsynonymous substitutions in Drosophila: mutation, selection or relaxed constraints?

Codon usage bias, the preferential use of particular codons within each codon family, is characteristic of synonymous base composition in many species, including Drosophila, yeast, and many bacteria. Preferential usage of particular codons in these species is maintained by natural selection acting largely at the level of translation. In Drosophila, as in bacteria, the rate of synonymous substitution per site is negatively correlated with the degree of codon usage bias, indicating stronger selection on codon usage in genes with high codon bias than in genes with low codon bias. Surprisingly, in these organisms, as well as in mammals, the rate of synonymous substitution is also positively correlated with the rate of nonsynonymous substitution. To investigate this correlation, we carried out a phylogenetic analysis of substitutions in 22 genes between two species of Drosophila, Drosophila pseudoobscura and D. subobscura, in codons that differ by one replacement and one synonymous change. We provide evidence for a relative excess of double substitutions in the same species lineage that cannot be explained by the simultaneous mutation of two adjacent bases. The synonymous changes in these codons also cannot be explained by a shift to a more preferred codon following a replacement substitution. We, therefore, interpret the excess of double codon substitutions within a lineage as being the result of relaxed constraints on both kinds of substitutions in particular codons.

Natural selection on synonymous sites is correlated with gene length and recombination in Drosophila.

Evolutionary analysis of codon bias in Drosophila indicates that synonymous mutations are not neutral, but rather are subject to weak selection at the translation level. Here we show that the effectiveness of natural selection on synonymous sites is strongly correlated with the rate of recombination, in accord with the nearly neutral hypothesis. This correlation, however, is apparent only in genes encoding short proteins. Long coding regions have both a lower codon bias and higher synonymous substitution rates, suggesting that they are affected less efficiently by selection. Therefore, both the length of the coding region and the recombination rate modulate codon bias. In addition, the data indicate that selection coefficients for synonymous mutations must vary by a minimum of one or two orders of magnitude. Two hypotheses are proposed to explain the relationship among the coding region length, the codon bias, and the synonymous divergence and polymorphism levels across the range of recombination rates in Drosophila. The first hypothesis is that selection coefficients on synonymous mutations are inversely related to the total length of the coding region. The second hypothesis proposes that interference among synonymous mutations reduces the efficacy of selection on these mutations. We investigated this second hypothesis by carrying out forward simulations of weakly selected mutations in model populations. These simulations show that even with realistic recombination rates, this interference, which we call the "small-scale" Hill-Robertson effect, can have a moderately strong influence on codon bias.

Intraspecific and interspecific variation at the y-ac-sc region of Drosophila simulans and Drosophila melanogaster.

A 2.2-kb region including the ac gene of Drosophila simulans has been sequenced. Interspecific divergence between Drosophila melanogaster and D. simulans was estimated as 0.0695 and 0.0558 for silent and for all sites, respectively. Estimated silent site divergence for the ac region is comparable to that estimated for other regions of the genome between these species, indicating that silent sites of the ac region are not under significantly stronger functional constraint. Intraspecific variation in both species was also investigated. Restriction-site and length polymorphism in the ac region of D. simulans has been investigated for 103 X chromosome lines sampled from three natural populations in Spain using eight four-cutter restriction enzymes. Neither restriction-site nor length variation was detected in the three populations surveyed. In D. melanogaster restriction-site and length polymorphism in all major transcription units of the y-ac-sc region (23.1-kb region) has been studied using four four-cutter restriction enzymes for 245 X chromosome lines sampled from 10 natural populations (seven from Europe, two from North America and one from Japan). Fourteen restriction-site and 28 length polymorphisms were detected. There was some indication of population subdivision for North American vs. European samples of D. melanogaster. The frequency spectrum of restriction-site polymorphisms in European populations was skewed toward rarer frequencies than predicted by the neutral theory. Comparison of silent site variation at this telomeric region with that in the Adh 5'-flanking region showed a reduced level of heterozygosity in the y-ac-sc region. Since interspecific silent divergence is not reduced in the y-ac-sc region as compared to other regions, the reduction in standing levels of variation at this telomeric locus in both D. simulans and D. melanogaster is most easily explained by a hitchhiking effect of linked selected substitutions.

Microarray-based capture of novel expressed cell type-specific transfrags (CoNECT) to annotate tissue-specific transcription in Drosophila melanogaster.

Faithful annotation of tissue-specific transcript isoforms is important not only to understand how genes are organized and regulated but also to identify potential novel, unannotated exons of genes, which may be additional targets of mutation in disease states or while performing mutagenic screens. We have developed a microarray enrichment methodology followed by long-read, next-generation sequencing for identification of unannotated transcript isoforms expressed in two Drosophila tissues, the ovary and the testis. Even with limited sequencing, these studies have identified a large number of novel transcription units, including 5' exons and extensions, 3' exons and extensions, internal exons and exon extensions, gene fusions, and both germline-specific splicing events and promoters. Additionally, comparing our capture dataset with tiling array and traditional RNA-seq analysis, we demonstrate that our enrichment strategy is able to capture low-abundance transcripts that cannot readily be identified by the other strategies. Finally, we show that our methodology can help identify transcriptional signatures of minority cell types within the ovary that would otherwise be difficult to reveal without the CoNECT enrichment strategy. These studies introduce an efficient methodology for cataloging tissue-specific transcriptomes in which specific classes of genes or transcripts can be targeted for capture and sequence, thus reducing the significant sequencing depth normally required for accurate annotation. Ovary and testis isotigs over 200 bp have been deposited with the GenBank Transcriptome Shotgun Assembly Sequence Database as bioproject no.PRJNA89451 (accession nos. JV208106­JV230865).

A method for estimating the numbers of synonymous and nonsynonymous substitutions per site.

A method for estimating the numbers of synonymous (Ks) and nonsynonymous (Ka) substitutions per site is proposed. The method is based on the Li's (J. Mol. Evol. 36:96-99, 1993) and Pamilo and Bianchi's (Mol. Biol. Evol. 10:271-281, 1993) method, but a putative source of bias is solved. It is proposed that the number of synonymous substitutions that are actually transitions or transversions should be computed by separating the twofold degenerate sites into two types of sites, 2S-fold and 2V-fold, where only transitional and transversional substitutions are synonymous, respectively. Kimura's (J. Mol. Evol. 16:111-120, 1980) two-parameter correcting method for multiple substitutions at a site is then applied using the overall observed synonymous transversion frequency to estimate both the numbers of synonymous transversional (Bs) and transitional (As) substitutions per site. This approach, therefore, also minimizes stochastic errors. Computer simulations indicate that the method presented gives more accurate Ks and Ka estimates than the aforementioned methods. Furthermore, the obtention of confidence intervals for divergence estimates by computer simulation is proposed.

K-Estimator: calculation of the number of nucleotide substitutions per site and the confidence intervals.

UNLABELLED: K-Estimator 4.5 is a Windows program that estimates the number of nucleotide substitutions per site (divergence) when comparing two aligned nucleotide sequences, both protein-coding and non-coding. Confidence intervals of the divergence estimates are obtained by Monte Carlo simulation. AVAILABILITY: The program is available for non-profit use via anonymous ftp at ftp.bio.indiana. edu/molbio/mswin. CONTACT: jcomeron@midway.uchicago.edu

An evaluation of measures of synonymous codon usage bias.

Synonymous codons are not generally used at equal frequencies, and this trend is observed for most genes and organisms. Several methods have been proposed and used to estimate the degree of the nonrandom use of the different synonymous codons. The estimates obtained by these methods, however, show different levels of both precision and dispersion when coding regions of a finite number of codons are under analysis. Here, we present a study, based on computer simulation, of how the different methods proposed to evaluate the nonrandom use of synonymous codons are affected by the length of the coding region analyzed. The results show that some of these methods are heavily influenced by the number of codons and that the comparison of codon usage bias between coding regions of different lengths shows a methodological bias under different conditions of nonrandom use of synonymous codons. The study of the dispersion of the estimates obtained by the different methods gives, on the other hand, an indication of the methods to be applied to compare values of codon usage bias among coding regions of equivalent length.

The molecular clock revisited: the rate of synonymous vs. replacement change in Drosophila.

Rates of synonymous and nonsynonymous substitution were investigated for 24 genes in three Drosophila species, D. pseudoobscura, D. subobscura, and D. melanogaster. D. pseudoobscura and D. subobscura, two distantly related members of the obscura clade, differ on average by 0.29 synonymous nucleotide substitutions per site. D. melanogaster differs from the two obscura species by an average of 0.81 synonymous substitutions per site. Using a method developed by Gillespie, we investigated the variance to mean ratio, or Index of Dispersion, R, of substitutions along the three species' branches to test the fundamental prediction of the neutral theory of molecular evolution, E(R) = 1. For nonsynonymous substitutions, the average R, Ra is 1.6, which is not significantly different from the neutral theory prediction. Only 5 of the 24 genes had significantly large Ra valves, and 12 of the genes had Ra estimates of less than one. In contrast, the Index of Dispersion for synonymous substitutions was significantly large for 12 of the 24 genes, with an average of R(s) = 4.4, also statistically significant. These findings contrast with results for mammals, which showed overdispersion of nonsynonymous substitutions, but not of synonymous substitutions. Weak selection acting to maintain codon bias in Drosophila, but not in mammals, may be important in explaining the high variance in the rate of synonymous substitutions in this group of organisms.

Genetic diversity of the human serotonin receptor 1B (HTR1B) gene.

We systematically and comprehensively investigated polymorphisms of the HTR1B gene as well as their linkage disequilibrium and ancestral relationships. We have detected the following polymorphisms in our sample via denaturing gradient gel electrophoresis, database comparisons, and/or previously published assays: G-511T, T-261G, -182INS/DEL-181, A-161T, C129T, T371G, T655C, C705T, G861C, A1099G, G1120A, and A1180G. The results of the intermarker analyses showed strong linkage disequilibrium between the C129T and the G861C polymorphisms and revealed four common haplotypes: ancestral (via chimpanzee comparisons), 129T/861C, -161T, and -182DEL-181. The results of association tests with schizophrenia were negative, although A-161T had a nominal P = 0.04 via ASPEX/sib_tdt. The expressed missense substitutions, Phe124Cys, Phe219Leu, Ile367Val, and Glu374Lys, could potentially affect ligand binding or interaction with G proteins and thus modify drug response in carriers of these variants. On average, the human cSNPs and differences among other primates clustered in the more thermodynamically unstable regions of the mRNA, which suggests that the evolutionary survival of nucleotide sequence variation may be influenced by the mRNA structure of this gene.